Dataset Information

Frequent detection of infectious xenotropic murine leukemia virus (XMLV) in human cultures established from mouse xenografts.

ABSTRACT:

Purpose

To investigate the frequency of xenotropic murine leukemia virus (MLV) presence in human cell lines established from mouse xenografts and to search for the evidence of horizontal viral spread to other cell lines.

Results

Six of 23 (26%) mouse DNA free xenograft cultures were strongly positive for MLV and their sequences had greater than 99% homology to known MLV strains. Four of five available supernatant fluids from these viral positive cultures were strongly positive for RT activity. Three of these supernatant fluids were studied to confirm the infectivity of the released virions for other human culture cells. Of the 78 non-xenograft derived cell lines maintained in the xenograft culture-containing facilities, 13 (17%) were positive for MLV, including XMRV, a virus strain first identified in human tissues. By contrast, all 50 cultures maintained in a xenograft culture-free facility were negative for viral sequences.

Methodology

We examined xenograft tumor cell lines from seven independent laboratories and 128 non-xenografted tumor cell lines. Cell line DNA was examined for mouse DNA contamination, and by 3 Taqman qPCR assays targeting the gag, env or pol regions of MLV. Sequencing was used for viral strain identification. Supernatant fluids were tested for reverse transcriptase (RT) activity.

Conclusions

Human cultures derived after mouse xenografting frequently contain and release highly infectious xenotropic MLV viruses. Laboratories working with xenograft-derived human cultures should be aware of the risk of contamination with potentially biohazardous human-tropic mouse viruses and their horizontal spread to other cultures.

SUBMITTER: Zhang YA

PROVIDER: S-EPMC3218386 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:One of the unique features of gammaretroviruses is that they contain an additional extended form of Gag, glyco-gag, which initiates in the leader sequence. MuLV glyco-gag, gPr80Gag, promotes retrovirus replication and disease progression. Although virtually all infectious MuLVs encode glyco-gag, XMRV (xenotropic murine leukemia virus-related virus) lacks the classical gPr80Gag sequence. We examined XMRV to determine if its leader sequence contains glyco-gag activity, whether the presence of conventional gPr80Gag affects replication of XMRV, and we describe the evolution of glyco-gag-deficient MuLVs in Mus. RESULTS:We introduced several mutations disrupting two putative but noncanonical glyco-gag proteins in the leader sequence region in XMRV and found that those mutations did not affect virus release nor susceptibility to the antiviral activity of hA3G (human APOBEC3G). A chimeric XMRV encoding the Moloney MuLV (M-MuLV) leader sequence (MXMRV) demonstrated that M-MuLV glyco-gag facilitated MXMRV release and increased infectivity. Infectivity assays with several cell lines showed that glyco-gag increases XMRV infectivity in all cell lines tested, but the level of this increase varies in different cell lines. Because MuLV glyco-gag counteracts mouse APOBEC3, we investigated whether M-MuLV glyco-gag enhances XMRV infection by counteracting human APOBEC3. Comparison of hAPOBEC3 isoforms expressed in different cell lines indicated that hA3B was the most likely candidate for a restrictive hA3. However over-expression of hA3B showed no enhanced restriction of infection by XMRV compared to MXMRV. Endogenous MuLVs in the sequenced mouse genome were screened for canonical glyco-gag, which was identified in two clades of xenotropic MuLVs (X-MuLVs) and ecotropic MuLVs, but not in other X-MuLVs or in any polytropic MuLVs. CONCLUSIONS:M-MuLV glyco-gag facilitates XMRV replication, and the leader sequence region in XMRV does not encode proteins equivalent to M-MuLV glyco-gag. The fact that the ability of glyco-gag to enhance XMRV infection varies in different cell lines suggests a glyco-gag sensitive restrictive factor that further reduces XMRV infectivity. The M-MuLV glyco-gag enhancement for XMRV replication is through a hAPOBEC3 independent mechanism. The absence of glyco-gag in MuLVs carried by western European mice suggests that loss of this sequence is a relatively recent event with limited subspecies distribution.